DESCRIPTION (adapted from the application) We previously identified the adapter protein Grb10 as an interacting partner for the IGF-I receptor (IGF-IR), showing that an over-expressed Grb10 was inhibiting IGF-I-induced but not insulin-induced mitogenesis in mouse embryo fibroblasts over-expressing either the IGF-IR or the insulin receptor (IR). With a two-hybrid screen using mGrb10 as a bait we isolated Nedd4, an ubiquitin protein ligase, as an interacting partner with Grb10. We could not detect ubiquitination of Grb10, but we have preliminary evidence of ubiquitination of the IGF-IR. We showed also that Grb10 is inhibiting IGF-I-induced differentiation of H19-7/IGF-IR rat hippocampal cells. Our previous work and our preliminary data suggest the hypothesis that Grb10 is a negative regulator of IGF-I signaling, acting probably through ubiquitination of the IGF-IR. To test this hypothesis we propose: To investigate the mechanism of inhibition of IGF-I-induced mitogenesis by Grb10, using mouse embryo fibroblasts over-expressing Grb10 in combination with either an over-expressed IGF-IR or over-expressed IR; To study the biological significance of the Grb10/Nedd4 interaction and the role of this complex in ubiquitination of the IGF-IR, using different cell lines overexpressing Grb10 in combination with different numbers of IGF-IR; To investigate the role of Grb10 in IGF-I-induced differentiation of H19-7/IGF-IR cells. These are immortalized rat hippocampal cells, where the data obtained in differentiation at 39 degrees C, will be compared with data obtained at 34 degrees C, where H19-7/IGF-IR cells can be tested for IGF-I-induced mitogenesis or survival. The significance of this project is not only in terms of a better understanding of the biological role of mGrb10 and its role in signaling from receptor tyrosine-kinases, but, in the long range, this study could yield valuable information for translational research. Considering the role of the IGF-IR in the establishment of the transforming phenotype, this grant proposal could also help us in designing better and more specific therapeutic agents, targeting specific components of a particular pathway without interfering with other signals necessary for the normal function of the cell.